WO2010030789A1 - Dispositif et procédé pour l'inhibition de l'activation du complément - Google Patents

Dispositif et procédé pour l'inhibition de l'activation du complément Download PDF

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Publication number
WO2010030789A1
WO2010030789A1 PCT/US2009/056524 US2009056524W WO2010030789A1 WO 2010030789 A1 WO2010030789 A1 WO 2010030789A1 US 2009056524 W US2009056524 W US 2009056524W WO 2010030789 A1 WO2010030789 A1 WO 2010030789A1
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Prior art keywords
antibody
complement
blood
properdin
subject
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PCT/US2009/056524
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English (en)
Inventor
Rekha Bansal
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Novelmed Therapeutics, Inc.
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Priority to US13/063,301 priority Critical patent/US20110160636A1/en
Publication of WO2010030789A1 publication Critical patent/WO2010030789A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
    • A61M1/3472—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate
    • A61M1/3486—Biological, chemical treatment, e.g. chemical precipitation; treatment by absorbents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3679—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by absorption
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54—F(ab')2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to complement activation. Particularly, the present invention relates to a method for inhibiting complement activation via an extracorporeal device.
  • Complement pathway is activated when blood comes in contact with an artificial surface of an extracorporeal device.
  • the cascade of events start and progress into forming anaphylatoxins and finally activating leukocytes, lymphocytes, and platelets. All these cell types are known to have receptors for anaphylatoxins, which cause cellular activation leading to pathological consequences.
  • the native C3 is converted into C3b and C3a.
  • the newly formed C3b interacts with properdin and factor B to form the C3 convertase, which cleaves additional C3 molecules into C3b and C3a and C5 molecules into C5b and C5a. Both C3a and C5a are potent anaphylatoxins.
  • C5b deposits onto cell surfaces and can initiate the formation of C5b-9 complexes.
  • Neutrophils, monocytes and platelets have receptors for anaphylatoxins C3a and C5a and therefore effectively activates these cell types.
  • T lymphocytes and mast cells can also be activated by anaphylatoxins.
  • the present invention relates to an extracorporeal device for inhibiting alternative complement pathway activation.
  • the extracorporeal device includes a support structure, an anti-complement antibody disposed on or within the support structure, and a first conduit for conducting blood to the anti-complement antibody.
  • the anti-complement antibody can bind to complement protein and remove the complement protein from the blood. Binding and removing the complement protein from the blood of a subject can inhibit the anti- complement alternative complement pathway activation in the blood of the subject.
  • the support structure can include a matrix that comprises at least one of agarose, cellulose, dextrin, polystyrene, polyethersulfone, polyvinyl difluoride, ethylene vinyl alcohol, polycarbonate, polyether, polyether carbonate, regenerated cellulose, cellulose acetate, polylactic acid, nylon, or polyurethane.
  • the anti-complement antibody can be coated on the support structure.
  • the antibody can include at least one of an anti-C3 antibody, anti-C3b antibody, anti-Ba antibody, anti-Bb antibody, anti-P antibody, anti-D antibody, anti-C5 antibody, anti- C5a antibody, anti-C6 antibody, anti-C7 antibody, anti-C8 antibody, and anti-C9 antibody.
  • the antibody can be raised in a mammal.
  • the antibody can also be monoclonal, polyclonal, recombinant, monospecific, bispecific, dimeric, humanized, chimeric, single chain, human, bispecific, truncated or mutated.
  • the antibody can be an IgG, F(ab')2, F(ab)2, Fab', Fab, scFv, truncated IgG, or recombinant antibody.
  • the blood contacted with the anti-complement antibody is incapable of activating the alternative complement pathway when returned to the subject.
  • the removal of the complement protein in the blood prevents activation of neutrophils, monocytes, basophils, lymphocytes, and platelets via the alternative pathway.
  • the anti-complement antibody can reduce the level of properdin in the blood.
  • the reduced levels of properdin in blood can decrease levels of C3a, C5a, Bb, C5b-9 as a result of decreased alternative complement pathway activation during extracorporeal circulation.
  • the reduced levels of properdin can also reduce cellular activation in blood from the subject following extracorporeal circulation.
  • the device can include a second conduit for returning blood to the subject.
  • the complement protein is removed from the blood returned to the subject.
  • the anti-complement antibody can be covalently adhered to a biocompatible polymer matrix.
  • the polymer matrix can be in the form of a membrane.
  • the support structure can also include the particulate polymer matrix.
  • the particulate polymer matrix can have reactive groups, such as an aldehyde, hydroxyl, thiol, carboxyl and/or amino groups that are capable of reacting with the antibody to adhere the antibody to the matrix.
  • the extracorporeal device can be coupled to at least one of an artificial heart-lung device or a hemodialysis unit such that blood flows through both the artificial heart lung device or hemodialysis unit and the extracorporeal device.
  • the present invention also relates to a method of inhibiting alternative complement pathway activation in a subject.
  • the method includes passing a bodily fluid of the subject through an extracorporeal device.
  • the device can include a support structure, an anti-complement antibody disposed on or within the support structure, and a first conduit for conducting bodily fluid of the subject to the anti-complement antibody.
  • the anti- complement antibody can bind to and remove complement protein in the bodily fluid.
  • the bodily fluid contacted with anti-complement antibody can then returned to the subject.
  • the bodily fluid can comprise whole human blood.
  • the support structure can include a matrix that comprises at least one of agarose, cellulose, dextrin, polystyrene, polyethersulfone, polyvinyl difluoride, ethylene vinyl alcohol, polycarbonate, polyether, polyether carbonate, regenerated cellulose, cellulose acetate, polylactic acid, nylon, or polyurethane.
  • the anti-complement antibody can be coated on the support structure.
  • the antibody can include at least one of an anti-C3 antibody, anti-C3b antibody, anti-Ba antibody, anti-Bb antibody, anti-P antibody, anti-D antibody, anti-C5 antibody, anti- C5a antibody, anti-C6 antibody, anti-C7 antibody, anti-C8 antibody, and anti-C9 antibody.
  • the antibody can be raised in a mammal.
  • the antibody can also be monoclonal, polyclonal, recombinant, monospecific, bispecific, dimeric, humanized, chimeric, single chain, human, bispecific, truncated or mutated.
  • the antibody can be an IgG, F(ab')2, F(ab)2, Fab', Fab, scFv, truncated IgG, or recombinant antibody.
  • the blood contacted with the anti-complement antibody is incapable of activating the alternative complement pathway when returned to the subject.
  • the removal of the complement protein in the blood prevents activation of neutrophils, monocytes, basophils, lymphocytes, and platelets via the alternative pathway.
  • the anti-complement antibody can reduce the level of properdin in the blood.
  • the device can include a second conduit for returning blood to the subject.
  • the complement protein is removed from the returned blood.
  • the anti-complement antibody can be covalently adhered to a biocompatible polymer matrix.
  • the polymer matrix can be in the form of a membrane.
  • the support structure can also include a particulate polymer matrix.
  • the particulate polymer matrix can have reactive groups, such as an aldehyde, hydroxyl, thiol, carboxyl and/or amino groups that are capable of reacting with the antibody to adhere the antibody to the matrix.
  • the present invention also relates to an extracorporeal system for inhibiting alternative complement pathway activation in a subject.
  • the system includes a support structure, an anti-complement antibody disposed on or within the support structure, a first conduit for conducting blood of a subject to the anti-complement antibody, and a second conduit for returning blood contacted with anti-complement antibody to the subject.
  • the anti-complement antibody can bind to and remove complement protein in the blood.
  • the support structure can include a matrix that comprises at least one of agarose, cellulose, dextrin, polystyrene, polyethersulfone, polyvinyl difluoride, ethylene vinyl alcohol, polycarbonate, polyether, polyether carbonate, regenerated cellulose, cellulose acetate, polylactic acid, nylon, or polyurethane.
  • the anti-complement antibody can be coated on the support structure.
  • the antibody can include at least one of an anti-C3 antibody, anti-C3b antibody, anti-Ba antibody, anti-Bb antibody, anti-P antibody, anti-D antibody, anti-C5 antibody, anti- C5a antibody, anti-C6 antibody, anti-C7 antibody, anti-C8 antibody, and anti-C9 antibody.
  • the antibody can be raised in a mammal.
  • the antibody can also be monoclonal, polyclonal, recombinant, monospecific, bispecific, dimeric, humanized, chimeric, single chain, human, bispecific, truncated or mutated.
  • the antibody can be an IgG, F(ab')2, F(ab)2, Fab', Fab, scFv, truncated IgG, or recombinant antibody.
  • the blood contacted with the anti-complement antibody is incapable of activating the alternative complement pathway when returned to the subject.
  • the removal of the complement protein in the blood prevents activation of neutrophils, monocytes, basophils, lymphocytes, and platelets via the alternative pathway.
  • the anti-complement antibody can reduce the level of properdin in the blood.
  • the reduced levels of properdin in blood can decrease levels of C3a, C5a, Bb, C5b-9 as a result of decreased alternative complement pathway activation during extracorporeal circulation.
  • the reduced levels of properdin can also reduce cellular activation in blood from the subject following extracorporeal circulation.
  • the anti-complement antibody can be covalently adhered to a biocompatible polymer matrix.
  • the polymer matrix can be in the form of a membrane.
  • the support structure can also include a particulate polymer matrix.
  • the particulate polymer matrix can have reactive groups, such as an aldehyde, hydroxyl, thiol, carboxyl and/or amino groups that are capable of reacting with the antibody to adhere the antibody to the matrix.
  • the extracorporeal system can be coupled with at least one of an artificial heart- lung device or a hemodialysis unit such that blood flows through both the artificial heart lung device or hemodialysis unit and the extracorporeal device.
  • Fig. 1 illustrates a properdin trimer attached to non-blocking and blocking anti- properdin monoclonal antibodies.
  • Fig. 2 illustrates a round bead matrix, which is coated with protein-G.
  • the monoclonal antibody MoAb 71"110 was conjugated to the matrix in a fully oriented form with binding regions exposed because protein-G is capable of orienting the monoclonal antibody.
  • the blood sample also shown is the blood sample.
  • Triangles represent the properdin molecules. Properdin from blood binds the antibody. The bound properdin can be eluted off the column.
  • Fig. 3 illustrates a device in accordance with the present invention before and after the blood has passed through. Properdin bound beads are shown in zoom.
  • Fig. 4 illustrates the blood with properdin being introduced into the "device”. As shown the blood coming out from the other end is free of properdin.
  • Fig. 5 compares the AP dependent hemolysis of human serum eluted off the three columns that differ in the way monoclonal antibody 7i ⁇ o is bound to the matrix. Three columns were prepared. The first column has sepharose beads 300 microns with monoclonal antibody 7i ⁇ o covalently linked via the linker chemistry. The second bead column contain the F(ab')2 fragment of the monoclonal antibody. In the third case, the beads are smaller in size - 160 microns. These beads were coated with covalently bound protein-G. The monoclonal antibody was cross linked to the protein G to acquire the correct orientation onto the bead.
  • Fig. 6 illustrates repetition of the experiment in Figure 5 but by using the third column. A small 2 ml column was set up and nearly 10 ml of the human serum was passed over the column to allow properdin depletion from serum.
  • Fig. 7 illustrates the absence of properdin in fractions that demonstrated lack of AP activation. Both positive and negative controls showed appropriate values. The monoclonal antibody added to control serum prevents complement activation and hence prevents properdin binding to C3b. the presence of free properdin in all fractions was measured.
  • Fig. 8 illustrates the presence of factor B to the same levels in all samples clearly suggesting that the device in accordance with the present invention does not affect the levels of factor B.
  • Fig. 9 illustrates the evaluation of efficacy of the 2 ml column. This experiment is a repeat of the previous experiment but with larger volume of human serum (82 ml). Nearly 100 ml of human serum was introduced into the cartridge at a flow rate of 1 ml/minute. Fractions of 1 ml size were collected over the course of 2 hours. Each fraction was tested for AP activity and properdin (Figure 10) levels. Measurement of AP activity is shown in this Figure. As shown, fraction #82 corresponds to the 82 ml of human serum lacks the AP activity. However, the AP activity appear to return at fractions around 85-88. Serum control with full activity is shown. Fractions near 100 or greater display the same lysis kinetic.
  • Fig. 10 illustrates the levels of properdin in column fractions.
  • Fig. 11 illustrates the results of the experiment performed in Fig. 9.
  • the column following serum elution was extensively washed with PBS and eluted with elution buffer.
  • properdin was the sole player in making serum inactive towards AP activation
  • the bound proteins were eluted off the column.
  • the eluted material was subjected to 6-18% gradient SDS-PAGE.
  • the blots were prepared and stained with anti-P, anti-B and only secondary antibody. As shown, there is no factor D or the artifact as the third blot showed no band in the absence of any primary antibody. However, the first blot shows properdin bands near 50K region.
  • the present invention provides a method of making and using an extracorporeal device for removing a complement protein from bodily fluids of a subject so that the bodily fluid loses the ability to activate complement pathways.
  • the bodily fluid is not limited to blood, but can include other bodily fluids, such as plasma and serum. Removal of complement proteins is performed using antigen- antibody interactions. Anti-complement antibodies are immobilized onto a solid support structure of the extracorporeal device and a bodily fluid, such as blood, can be passed through the device.
  • a bodily fluid such as blood
  • the anti-complement antibody of the extracorporeal device can cause a complete/partial depletion of a target complement protein from the bodily fluid and can reduce in the subject or patient being treated: alternative complement pathway activation, complexes of antigen and antibody, levels of C3a/C5a (compared to C3a/C5a levels present in a subject or patient at the start of the extracorporeal procedure) the ability to make C5b-9 complexes, the levels of complement dependent activation of neutrophils, monocytes, and platelets, the levels of cytokines, TNF alpha, and platelet-monocyte conjugate, bleeding complications as well as inflammatory responses.
  • the anti-complement antibody can be monoclonal, polyclonal, monospecific, bispecific.
  • the anti-complement antibody can be murine, mammalian, fully human, recombinant, chimeric, mutated or truncated.
  • the anti-complement antibody can be a detection antibody or blocking antibody.
  • the antigen binding fragments of the antibody can also be used. In essence, any peptide, protein, or amino acid motifs that can bind the complement protein of interest are within the scope of the present invention.
  • variable regions of antibodies are conserved among IgG, F(ab)2, F(ab')2, Fab', Fab, scFv, recombinant, human, and truncated proteins - these can be immobilized onto the solid support to remove the complement proteins from body fluids.
  • the anti-complement antibody binding support structure can include a polymer matrix that has a substantial number of reactive groups, such as aldehyde, hydroxyl, thiol, carboxyl or amino groups, which can be activated for coupling the anti-complement antibody to the supportive structure.
  • a polymer support matrix may include natural carbohydrates, such as agarose, cellulose or dextran or synthetic polymers including polystyrene, poly ethersulf one, PVDF, ethylene vinyl alcohol, polycarbonate, polyether, polyether carbonate, regenerated cellulose, cellulose acetate, polylactic acid, nylon, or polyurethane.
  • the physical shape of the matrix can be beads, fibers, tapes, filters.
  • the anti-complement antibody can be conjugated to the matrix by direct chemical linking, lipohillic moieties, or by other proteins known in the art.
  • Proteins used to bind the anti-complement antibody to the matrix include proteins G, A, L and those that can mediate the binding of the matrix to the antibody without the loss of the antigen binding ability of the antibody to the proteins in fluid.
  • the antibody-bound matrix can be contained in a column or housing that allowS fluid flow.
  • the material of the column can be an inert material.
  • the column can have an inlet conduit and an outlet conduit and a fritted disc.
  • the column can also withstand the flow rate commonly used in extracorporeal procedures.
  • the column can also contain a valve to prevent the flash back of the forward moving blood.
  • the extracorporeal device can be used alone (i.e., an extracorporeal circuit) to remove complement protein from blood before an extracorporeal circulation or procedure is begun. Removal of complement protein from the blood prior to an extracorporeal procedure such as cardio pulmonary bypass (CPB) is advantageous because the flow rate used for the extracorporeal device of the present invention may not be compatible with the flow rates observed during blood circulation in an extracorporeal CPB circuit.
  • CPB cardio pulmonary bypass
  • the extracorporeal device can also be used concurrently with the extracorporeal circuit.
  • the device should be connected between the patient and the extracorporeal circuit in such a way that blood from patient should flow into the device prior to coming in contact with the extracorporeal circuit.
  • the extracorporeal device can include a substrate -bound anti -properdin (i.e., anti-P) antibody, which removes properdin from the blood.
  • a substrate -bound anti -properdin (i.e., anti-P) antibody which removes properdin from the blood.
  • the blood returning to the patient becomes devoid of properdin, which is critical for alternative complement pathway (i.e., AP) activation.
  • an anti-P monoclonal antibody that specifically blocks the AP can be immobilized onto large beads using protein-G coated sepharose B via chemical cross linking. The anti-P conjugated beads can then be incubated with whole human blood, plasma or serum.
  • the anti-P conjugated beads incubated with the whole human blood, plasma, or serum can extract properdin by specific binding on properdin functional site and properdin will be removed from the blood, plasma, or sera.
  • the extracorporeal device allows a sample of blood, plasma and sera to be safely rotated through an extracorporeal circuit with exposed foreign surfaces.
  • the beads to which the anti-P antibody is conjugated can be large enough to allow flow of cells through the extracorporeal device without shear. Because shear forces can cause cellular lysis, it is important to determine the size of beads appropriate for cells to pass through. CELLTHRUBIGBEADS (Sterogene Bioseparations, Inc., Carlsbad, Calif., USA) have been routinely used for such applications.
  • Anti-P can be conjugated directly to the beads but the binding efficiency of the anti-P may be reduced because of lack of proper orientation of the antibody onto the beads.
  • a support matrix with reactive functional groups such as, but not limited to, aldehyde, hydroxyl, thiol, amino or carboxyl groups, available for protein coupling can be used to promote anti-P antibody coupling to the matrix.
  • the anti-P can be immobilized by binding the anti-P to a cellulose support matrix, such as a regenerated cellulose hollow fiber membrane, which is used in hemodialyzers.
  • a cellulose support matrix such as a regenerated cellulose hollow fiber membrane
  • Cellulose contains abundant hydroxyl groups, which can be activated with sodium metaperiodate, thus oxidizing them to aldehyde groups.
  • Anti-P can be coupled to aldehyde groups of the cellulose by reductive amination.
  • Aldehyde derivatization of other supports such as polystyrene, polyethersulfone, PVDF, ethylene vinyl alcohol, polycarbonate, polyether, polyethercarbonate, polylactic acid, nylon, or polyurethane can be performed with formaldehyde or glutaraldehyde using standard chemical reactions (reviewed in Affinity Techniques, Methods in Enzymology Vol. 19A).
  • CELLTHRUB IGB EAD 300-500 micron beads (Sterogene Bioseparations, Inc., Carlsbad, Calif., USA) allowed blood passage through a packed column.
  • Anti-P can be immobilized onto the novel aldehyde activated 4% agarose beads (300-500 micron particles)(e.g., LS Activated CELLTHRUBIGBEAD (Sterogene Bioseparations, Inc., Carlsbad, Calif., USA)) at 5 mg/ml following the manufacturer's directions.
  • LS Activated CELLTHRUBIGBEAD Steogene Bioseparations, Inc., Carlsbad, Calif., USA
  • Human anti-P derivatized 300-500 micron CELLTHRUB IGBEADs can be packed into 5 ml columns and perfused with 500 ml of fresh heparinized human blood. The flow rate that gives maximum retention of properdin and no shear of cells can then be determined. The ratio of volume of cartridge to the volume of blood can then be estimated based on the data generated. High flow rate is required in light of its use in a cardio pulmonary bypass (CPB) circuit. The effluents can be tested for red blood cell (RBC) hemolysis. The whole profile of cell differential can also be determined.
  • the device of this invention can be optimized for two different settings. In one setting, the device can be used without the CPB circuit being connected.
  • a subject or patient can be connected to the device using catheters/tubing and a pump can be used to allow blood passage through the device.
  • the effluent blood can be free of properdin where beads with anti-P coating is used. Cells in effluent blood should have the same profile as of the incoming blood (blood coming into the device). In optimizing this device, flow rate of blood does not have to match the flow rate used in CPB circuit.
  • the device can also be used as a connector between the CPB circuit and the patient. Blood from the patient can flow into the device. The device can bind properdin in blood and pump the blood into the circuit. Such device would be ideal if flow rate of the device and CPB circuit is kept similar to avoid shear of RBCs and platelets. [0054] Other extracorporeal settings can also use such a device since it is practically a complement protein removal system. Similar devices can also be made by coating the beads/matrix with anti-factor D, anti-factor C5 antibodies, andti-C5a antibodies and others. In case of anti-C5a, the device can also provide a means of continuously removing C5a from blood.
  • the extracorporeal device can also be used in the extracorporeal applications, such as dialysis, plasmapheresis, extracorporeal membrane oxygenation, hemodialysis, hemofiltration, open-heart surgery, and organ transplantation.
  • Extracorporeal circulation is in part pathogenic because blood contact with the artificial circuits generates an inflammatory response and complement and other pathways relevant to blood are activated. Following the activation of complement, an intense cellular inflammatory response sets in causing a pathology, which leads to complications that arise following the extracorporeal circulation.
  • the main mechanism by which extracorporeal circulation of blood leads to morbidity and mortality is by producing anaphylatoxins C3a and C5a along with sC5b-9 complexes.
  • All components C3b, factor B, factor Bb, factor P, Factor C5, Factor D, Factor C6, Cl, C8, and C9 can be removed by affinity adsorption, utilizing as the adsorbent antibodies to these proteins or other specific chemical adsorbents, such as those that specifically bind the complement proteins.
  • Anti-complement antibodies bound to the matrix can remove specific proteins from blood because antibodies are known to be highly target specific.
  • Antibodies may be "detection or non-blocking” antibodies which detect the presence of the protein in a sample.
  • Antibodies may be "blocking" antibodies, which bind to the protein at a specific site involved in function.
  • anti-C5 antibodies have been produced that prevent factor C5 cleavage into C5a and C5b (e.g., see PCT/US08/57468).
  • Anti-P antibodies have been produced that prevent properdin binding to C3b (See PCT/US08/68530).
  • Both blocking and non-blocking antibodies can be used for removing proteins from a fluid. Both products can remove the specific protein from the fluid and therefore can be used to lower the concentration of the protein in fluid. There are major differences in these two approaches. When non-blocking antibodies are used for removing the specific protein, the functional site of the protein is not blocked and a fully functional protein remains bound to the matrix. When blocking antibodies are used for removing the specific protein, the functional site of the protein is blocked by the antibody to which the protein binds. The difference in the two approaches is highly significant with regards to generating an extracorporeal device. In the first approach, the device after coming in contact with the fluid will have the non-blocking antibody attached to the fully functional protein.
  • a non-blocking anti -properdin monoclonal antibodies when bound to a matrix in a device will remove properdin by binding to a non-active regions on properdin.
  • a blocking anti-properdin monoclonal antibodies when bound to the matrix in a device will remove properdin by binding to the functionally active regions on properdin.
  • the use of a blocking anti-complement antibody is preferred because the complement protein is neutralized as it is being removed from circulation.
  • a non-blocking properdin antibody will remove properdin that while in the device will be capable of activating the complement pathway and could accumulate and participate in alternative pathway C3 convertase formation.
  • the device of this invention can be prepared using blocking monoclonal antibodies especially those antibodies that are raised against intact, fragments, and fusion proteins derived from complement cascade.
  • the utilization of extracorporeal adsorption of properdin or any other complement protein the depletion of which can block the AP pathway is provided by this invention. Such adsorption can lead to marked reduction in the levels of that protein, thus would result in down regulation of the inflammatory responses during extracorporeal procedures.
  • the device of the present invention thus provides a non invasive means of reducing AP activation in a human and has a significantly larger quantitative effect on additional factors that are involved in the etiology and pathogenesis of inflammation. For example, reduction of complement activation will also inhibit activation of cells that are part of the inflammatory cascade.
  • the immuno-affinity adsorption of the antibody can be improved by immobilizing the antibody to the Staphylococcal Protein A.
  • Staphylococcal Protein A a recombinant Staphylococcal Protein A or Staphylococcal Protein A component, or other synthetic peptides of Staphylococcal Protein A may be utilized, as may Protein G or its components.
  • the terms "Protein A” and "Protein G" include all such variations.
  • fragments of antibodies when used in the present invention as affinity adsorbents, they can be produced by enzymatic (e.g., papain or pepsin) digestion of the intact antibody to produce Fab, (Fab')2, or FV antigen-binding fragments, or they can be produced by other methods known to those skilled in the art for the synthesis of peptides, such as solid phase synthesis (R. A. Houghten, Proc. National Academy of Science USA, Vol. 82, August 1985, pp. 5131-35; R. E. Bird et al., Science, Vol. 242, 1988, pp. 423-42).
  • the use of fragments, rather than intact antibodies, as the affinity adsorbent may increase the adsorption capacity and reduce side effects that may be associated with the Fc non-antigen binding part of the antibody molecule.
  • anti-P antibody that blocks the AP activation.
  • the anti-P antibody is described in PCT/US08/68530. This antibody binds properdin and blocks properdin function. Properdin plays a role in AP activation and therefore, blockade of properdin function inhibits the AP.
  • Fig. 1 shows a trimer of properdin monomer.
  • Anti-P binds the TSR-I, which is represented in the Figure as corners of the trimer. Based on the molar ratio of anti-P to properdin, the model shown perfectly fits the anti-P used. This particular model also shows that if anti-P is immobilized onto the matrix and correctly oriented, it should bind the trimer and retain it onto itself.
  • the blood/plasma samples passing through should become depleted of properdin. Since properdin plays a critical role AP, the blood and plasma should not activate the AP during blood transit through the extracorporeal circuit.
  • non-blocking anti-P which binds properdin but does not inhibit the AP activation is used, such antibodies will remove properdin from solution as shown by the binding of anti-P to the properdin trimer but the bound properdin would still be functional and would significantly activate the AP as more blood passes through the device.
  • trimer While properdin exists in all forms monomer, dimer, trimer, and tetramer, we will only be using the term trimer for convenience. The corners of the trimer will bind C3b and make an active C3 convertase in situ to allow AP activation to proceed. The device can then become a rich source of concentrated C3 convertase.
  • the anti-P is the one that blocks the AP and blocks the functionally important sites on the properdin molecule.
  • Bead matrix (CELLTHRUB IGB EADS (Sreogene Corporation) or any bead with large diameter of nearly 300 microns) uncoated or coated with protein G is incubated with the anti-P monoclonal antibody to generate anti-P coated beads.
  • Whole heparinized blood containing functional AP complement proteins is passed through the device.
  • the anti-P coated onto the beads bind properdin from plasma. The flow through should have no AP activity.
  • Fig. 3 illustrates two columns. The first column only has anti-P conjugated to the beads. The second column illustrates zoomed-out version of anti-P coated neads with retained properdin. An inset shows the zoom-in portion of the single bead with retained properdin.
  • Fig. 4 shows that the blood that has been through the device is depleted off properdin. The outlet from the device is being poured into the container. The trimer triangles are missing from the flow through.
  • the diluted serum pool was assayed by erythrocyte hemolysis assay.
  • 100 ⁇ l of the diluted serum was mixed with rabbit erythrocytes. The mixture was incubated at 37°C in a temperature controlled ELISA plate. The lysis of cells was monitored at 700 nm over time. As shown the control serum shows complete lysis by the serum indicating AP activation. Both columns with CELLTHRUBIGBEADS also lysed the rabbit erythrocytes suggesting that human serum samples were nearly as potent as the untreated controls. The slight delay in lysis may only reflect dilution effect from the column and we conclude based on these data that both CELLTHRUBIGBEADS did not work.
  • a column was prepared to evaluate the specificity of the substrate-bound monoclonal antibody on the protein-G matrix. Similar to example 4 above, the protein-G column was obtained from Thermo Scientific (Pierce Protein G IgG Plus Orientation Kit, cat# 44990). The monoclonal antibody was conjugated according to the manufacturer's instructions. The monoclonal antibody conjugated beads (2 ml) were placed in the polypropylene column. The column was washed phosphate buffer saline, pH 7.4. The human serum (5 ml) was placed on the column to allow its passage through the column. A total of five 1 ml fractions were collected. Each fraction was diluted with AP buffer and subjected to rabbit erythrocyte hemolysis assay.
  • Properdin is known to bind C3b with high affinity.
  • the presence of properdin was detected with anti-properdin antibody (P#2) from Quidel Corporation.
  • This primary antibody was diluted 1:2000 in blocking buffer before application.
  • the secondary antibody we used was an HRP-conjugated goat-antimouse monoclonal antibody. This antibody was also used at 1:2000 dilution.
  • Fig. 7. [0071] To determine if the loss of other proteins was contributing to the observed inhibition of AP activation, we measured the levels of factor B using an ELISA assay. In this assay, a monoclonal to factor B was coated onto the ELISA wells (2 ⁇ g/50 ⁇ l/well). Following blocking the wells were incubated with serum from various fractions. The bound factor B was detected with a polyclonal to factor B (American Qualex). The amount of factor B was determined using ordinary methods of detection. As shown in Fig. 8, levels of factor B were the same in all fractions suggesting no loss of factor B
  • a column was prepared to evaluate the specificity of the substrate-bound monoclonal antibody on the protein-G matrix. Similar to example 4 above, the protein-G column was obtained from Thermo Scientific (Pierce Protein G IgG Plus Orientation Kit, cat# 44990). The monoclonal antibody was conjugated according to the manufacturer's instructions. The monoclonal antibody conjugated beads (2 ml) were placed in the polypropylene column. The column was washed phosphate buffer saline, pH 7.4. The human serum (5 ml) was placed on the column to allow its passage through the column. A total of five 1 ml fractions were collected. Each fraction was diluted with AP buffer and subjected to rabbit erythrocyte hemolysis assay.
  • Properdin is known to bind C3b with high affinity.
  • the presence of properdin was detected with anti-properdin antibody (P#2) from Quidel Corporation.
  • This primary antibody was diluted 1:2000 in blocking buffer before application.
  • the secondary antibody we used was an HRP-conjugated goat-antimouse monoclonal antibody. This antibody was also used at 1:2000 dilution.
  • Fig. 7. [0073] To determine if the loss of other proteins was contributing to the observed inhibition of AP activation, we measured the levels of factor B using an ELISA assay. In this assay, a monoclonal to factor B was coated onto the ELISA wells (2 ⁇ g/50 ⁇ l/well). Following blocking the wells were incubated with serum from various fractions. The bound factor B was detected with a polyclonal to factor B (American Qualex). The amount of factor B was determined using ordinary methods of detection. As shown in Fig. 8, levels of factor B were the same in all fractions suggesting no loss of factor B
  • the column was extensively rinsed with phosphate buffered saline.
  • the column was eluted with an IgG Elution buffer (product # 21004) and the collected eluate was analyzed by western blot assay.
  • a gradient 8-16% SDS-PAGE was run and the gel was transblotted onto a PVDF membrane using standard Western blotting techniques.
  • One blot was probed with anti-properdin polyclonal antibody, the second one was probed with an anti-factor D polyclonal antibody and the third was probed without the primary antibody. All three blots were treated with a common secondary antibody - rabbit anti-goat polyclonal at 1:2000 dilution.
  • Fig. 11 Shown in Fig. 11 are blots where properdin was identified using antibodies to properdin.
  • panel A is shown properdin
  • Panel B is from Factor D antibody probed blot and Panel is a control.
  • the eluted protein from the "Device” is the properdin and not factor B. the band shown is not an artifact of the secondary antibody as the panel C shows no bands.

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Abstract

L'invention porte sur un dispositif extracorporel pour l'inhibition de l'activation de la voie alterne du complément, le dispositif comprenant une structure de support, un anticorps anti-complément placé sur la structure ou à l'intérieur de la structure de support et une première canalisation pour l'acheminement de sang du sujet vers l'anticorps anti-complément. L'anticorps anti-complément peut se fixer à la protéine du complément et éliminer du sang la protéine du complément.
PCT/US2009/056524 2008-09-10 2009-09-10 Dispositif et procédé pour l'inhibition de l'activation du complément WO2010030789A1 (fr)

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USD952157S1 (en) 2020-06-19 2022-05-17 Qualigen Inc. Whole blood treatment cartridge

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US9216386B2 (en) * 2009-03-17 2015-12-22 Marv Enterprises, LLC Sequential extracorporeal treatment of bodily fluids
US20150027950A1 (en) * 2012-03-27 2015-01-29 Marv Enterprises, LLC Treatment for atherosclerosis
US10151749B2 (en) * 2015-08-05 2018-12-11 Alfaisal University Method and kit for the detection of microorganisms
WO2017127761A1 (fr) 2016-01-20 2017-07-27 Vitrisa Therapeutics, Inc. Compositions et procédés pour inhiber le facteur d
WO2018136827A1 (fr) 2017-01-20 2018-07-26 Vitrisa Therapeutics, Inc. Compositions à boucle en épingle à cheveux et procédés pour inhiber le facteur d
US20190247560A1 (en) * 2018-02-13 2019-08-15 Gambro Lundia Ab Extracorporeal devices and methods of treating complement factor related diseases

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USD952157S1 (en) 2020-06-19 2022-05-17 Qualigen Inc. Whole blood treatment cartridge

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